Understanding the enzymatic mechanisms of DNA replication is an important problem at the foundation of molecular biology. The overarching goal of our studies is to develop a quantitative model of DNA replication that will accurately recapitulate the catalytic properties of the enzymes (helicase, polymerase, primase) and the functional coupling between them during the reactions of DNA unwinding, synthesis, and priming. Our work will advance the field of DNA replication by providing detailed insights into how helicase and polymerase work together and how leading and lagging strand synthesis are coupled. We propose studies of replicative enzymes of the bacteriophage T7 and the human mitochondria. Phage T7 encodes one of the simplest replication systems that provide a model for human mitochondrial DNA replication as well as more complicated systems. Defects in helicase and polymerase functions are associated with diseases such as cancer, premature ageing, and neuromuscular disorders. A detailed understanding of the enzymatic mechanisms of helicase and polymerase mechanisms will enable development of therapeutics for such diseases. Quantitative transient state and single molecule kinetics studies allowed us to discover new mechanisms and synergies between the T7 replicative enzymes. A major goal with the T7 studies is to understand the collaborative coupling between the replicative enzymes during leading and lagging strand DNA synthesis. A major goal of the mitochondrial studies is to reconstitute replication in vitro and characterize select disease- causing mutants of Twinkle. This proposal builds upon our characterization of T7 and mitochondria replication from the last grant cycle to address key questions that arose from prior results. How helicase and polymerase are physically and functionally coupled at the replication fork? What is the base pair stepping mechanism of the helicase-polymerase? How does collaborative coupling affect DNA synthesis fidelity (misincorporation, proofreading)? What is the mechanism of lagging strand DNA synthesis? How do specific disease-related point mutations in Twinkle change its function compared to wild-type? What is the role of the strand exchange activity of Twinkle? These fundamental questions on DNA replication will be addressed in the following specific aims: 1) Investigate coupling between the activities of helicase and polymerase during leading strand DNA synthesis. 2) Investigate mechanisms of lagging strand DNA synthesis. 3) Investigate mechanisms of wild-type and mutant helicase Twinkle and polymerase.